DE3734923C1 - Process for the preparation of a sterile plasma protein solution containing fibrinogen and coagulation factor XIII - Google Patents

Description

The invention relates to the method specified in claim 1 for producing a sterile plasma protein solution, the fibrinogen and the Coagulation factor XIII contains and especially as Component of a fibrin glue is suitable, from one with citrate stabilized human blood plasma. Claim 2 relates to a Organization of the procedure.

A number of different methods are known from the literature the fibrinogen production from plasmas known, of which most variants the one described by Cohn Use ethanol precipitation (Journal of the American Society 72 (1950), 564-574). A well known development of this The procedure is from Blombäck u. a. has been described (archive for Kemi 10 (1956), No. 29, 415-443).

Blood plasma fractions have a high risk of becoming infectious be. This risk particularly affects the transfer of Hepatitis B and Hepatitis Non-A / Non-B and more recently the HIV (human immunodeficiency virus).

Fibrinogen and the Cohn I fraction are among the so called "high risk" preparations in relation to the  Hepatitis risk (J.H. Hoofnagle et al .: J. Lab. Clin. Med. 88 (1976), 102; W. Doleschel et al. a .: Wiener Klin. Wschr. 89 (1977), 383).

In the USA, therefore, the clinical use of fibrinogen preparations, which are made from large plasma pools are prohibited.

Since diagnostic measures do not succeed, hepatitis-proof To produce blood products from plasma pools, are various methods for the sterilization of blood components has been developed. Pasteurization (10 h / 60 ° C) is successfully used for albumin and is through the use of stabilizers such as amino acids and Mono- or oligosaccharides and sugar alcohols since some years more sensitive to sterilization Plasma proteins such as coagulation factors II, VIII and XIII have been described (EP-B 00 18 561). The effectiveness of Pasteurization in the presence of these stabilizers is still cannot be estimated and is currently being reviewed.

The method of cold sterilization described by LoGrippo consists in the combined treatment of human plasma with β- propiolactone and UV radiation (GA LoGrippo and H. Hayashi: Henry Ford Hosp. Med. J. 21 (1973), 181; GA LoGrippo and FW Hartmann : Bibl. Haematol. 7 (1958), 225).

The sterilization of an already purified fibrinogen fraction with β- propiolactone / UV leads to fibrinogen products which can no longer be clotted (R. Kotitschke and W. Stephan: Proteins and Related Subjects, Vol. 28, Protides of Biological Fluids. Peeters H., 28th Colloquium 1980, Verlag Pergamon-Press, Oxford, New York-Toronto-Paris, 333-337; H. Brunner and others: Negotiations of the German Society for Inn. Med. 79, pp. 1130-1134 (1983)); W. Doeschel and W. Auerswald: Pharmacology 2 (1969), 1).

EP-B 00 14 333 describes a process for producing the plasma fractions, fibrinogen, a prothrombin complex containing the coagulation factors II, VII, IX and X (PPSB factors), antithrombin III and a solution of storage-stable serum proteins from a blood plasma stabilized with citrate , which was treated with β- propiolactone and UV radiation. According to this method, fibrinogen is produced from a citrate plasma pool which has been treated with β- propiolactone and UV-irradiated by adsorption on colloidal silica and subsequent elution. This fibrinogen is unsuitable as a component for a fibrin glue, since such a fibrinogen contains no factor XIII. In order for a fibrinogen preparation to meet the requirements for a fibrinogen that is suitable for fibrin bonding, it must be enriched with an F.XIII concentrate.

In the biological fibrin glue system, the through Ca ions and thrombin activated coagulation factor XIII (F.XIII) the individual fibrin polymers cross-linked so that an insoluble, firm and elastic fibrin clot arises.

(H. P. Spängler and F. Braun: Basics of fibrin bonding in of operative medicine, ed. H. P. Spängler and F. Braun: edition medicine. Weinheim; Deerfield Beach, Florida. Basel 1983).

The importance of the F.XIII content of the fibrin glue system has been demonstrated in skin adhesions in vivo (H. P. Spängler u. a .: Vienna. Clin. Weekly 85, 827 (1973)).  

Process for the production of sterile F.XIII concentrates from human plasma are very expensive, which is why F.XIII concentrates also preferably from human placents be produced (CA-PS 9 57 943). The F.XIII concentrate Production from plasma from which the cryoprecipitate has been removed is by Winkelman u. a. described (Thrombosis and Haemostasis 55, 3 (1986), 402-405). Solutions that reduce human coagulation factor XIII must contain to avoid the transmission of viruses be sterilized (EP-B 00 37 078).

The present invention was based on the object human plasma is a sterile and stable plasma protein solution to produce both the coagulation factor XIII and Fibrinogen in an amount concentrated in relation to the plasma contains and particularly suitable as a fibrin glue component is.

This object is achieved in that the blood plasma stabilized with citrate is treated with β- propiolactone at a pH of 7.2 in a concentration range from 0.2 to 0.35% by volume, based on the plasma used and irradiated with ultraviolet light (the UV dose is 4 × 20 watts at a distance of 1 cm with a layer thickness of the irradiated plasma of 0.15 mm and a flow rate of 20 l / h and 700 RPM (rotation of the UV tube per minute) ), then the coagulation factors II, VII, IX and X by adsorption on protein adsorbing anion exchangers, namely cross-linked dextrans or cellulose carrying diethylaminoethyl groups in an amount of 0.5-3 g per 1 kg plasma, followed by a precipitation with Ethanol to a final concentration of 9 vol .-% at -3 ° C, the precipitate is centrifuged and dissolved in a citrate buffer at a pH of 6.35 and 37 ° C with stirring, the protein value of the fibrinogen solution with sodium citrate solution to 13.3 g / l and additional accompanying proteins from 1 kg of fibrinogen solution with 260 ml of ethanol, 240 ml of glycine citrate buffer and 1500 ml of sodium citrate solution, the precipitate is centrifuged off and the fibrinogen and the coagulation factor from the supernatant XIII precipitates by adding ethanol to a final concentration of the solution of 9% by volume at -3 ° C., dissolves the precipitate at a pH of 6.35 and 37 ° C. with stirring in a citrate buffer to the desired fibrinogen concentration and filter the solution through a filter clarifier and a Millipore sterile filter.

If the fractionation of a β- propiolactone / UV-treated plasma is carried out in this way without prior separation of the coagulation factors II, VII, IX and X, a plasma fraction is obtained which contains the fibrinogen and the coagulation factor XIII in an unstable form, that is to say that products thus gelled and curdled so that further processing is not possible.

The process according to the invention turns a with Citrate stabilized human blood plasma to a clotting-active, Hepatitis-safe and therapeutically applicable plasma protein solution get the fibrinogen and the coagulation factor XIII in plasma enriched Takes shape. This plasma protein solution is particularly as Component of a fibrinogen adhesive suitable.

The table below shows the properties of two plasma protein solutions prepared by the process according to the invention, their protein concentration depending has been set differently from the intended use.  

table

The invention is illustrated by the example below explained.

Embodiment

60 blood plasma bags with freshly frozen citrate plasma with one Total weight of 37.2 kg were overnight at + 8 ° C leave. The pouches were then made using a scalpel opened under the laminar flow and the frozen plasma in a kettle with a heating jacket at 37 ° C with stirring inside Defrosted for 1 hour.

The plasma pool was cooled to about 10 ° C. in about 45 minutes. 92.5 ml of β- propiolactone (0.25%) was added dropwise to the plasma with stirring. After the pH had reached 7.2, it was kept constant at room temperature for 1 h by adding 1 N NaOH. 1 l of 1 N NaOH was consumed. The plasma treated with β- propiolactone was treated with a standard UV device with a UV dose of 4 × 20 watts at a distance of 1 cm with a layer thickness of the irradiated plasma of 0.15 mm and a flow rate of 20 l / h and 700 RPM ( Rotation of the UV tube per minute) exposed.

1.5 g of DEAE-Sephadex A-50 were used per 1 kg of plasma Adsorption of the PPSB factors used. After a stir from 1 h the Sephadex A-50 settled overnight. At the  the next day was the plasma standing over the sediment sucked off with the help of a riser pipe and a pump.

The pH of the plasma was adjusted to 7.2 and that Plasma cooled to 0 ° C. 96% ethanol was added Stir to a final concentration of 9% added. The Temperature was lowered to -3 ° C, the precipitation was 14 h. The precipitate was removed by centrifugation at a Centrifuge temperature of -15 ° C and one Centrifugation time of 1 l / min. at 11,000 rpm with one WKF centrifuge separated.

The precipitated plasma protein fraction (350 g precipitation) was with 1200 ml of a citrate buffer at pH 6.35 and + 37 ° C dissolved with stirring within 45 minutes.

A was used to adjust the protein value to 13.3 g / l Na citrate solution pH 6.35 (0.055 M citrate) was used. Per 1 kg Fibrinogen solution was used for the precipitation of Accompanying proteins a glycine citrate buffer pH 6.35 and Ethanol used in the following composition: 260 ml Ethanol, 240 ml glycine citrate buffer and 1500 ml Citrate solution. After a precipitation time of approx. 1 h Precipitated centrifuged and from the supernatant Fibrinogen and factor XIII by adding ethanol to precipitated to a final concentration of 9% at -3 ° C. The Precipitation was removed with a solution buffer at + 37 ° C Stir to the desired fibrinogen concentration dissolved.

The fibrinogen solution was first passed through a filter clarifier and then through a Millipore sterile filter filtered. The sterile filtered solution was used until stored for further use at -80 ° C frozen.  

The method according to the invention is schematic below shown:  

Schematic representation of the preparation of the sterile plasma protein solution (fibrinogen and F.XIII)

Claims (2)

1. A process for the preparation of a sterile and stable plasma protein solution, which contains fibrinogen and the coagulation factor XIII, from a human blood plasma stabilized with citrate, from which the coagulation factors II, VII, IX and X are separated by adsorption adsorbing protein and the separation of further Accompanying proteins are produced by fractional precipitation with ethanol, characterized in that the blood plasma stabilized with citrate is mixed with β- propiolactone in a concentration range from 0.2 to 0.35% by volume, based on the plasma used, and at a pH value of 7.2 treated and irradiated with ultraviolet light, then the coagulation factors II, VII, IX and X by adsorption on protein adsorbing anion exchangers, namely cross-linked dextrans or cellulose carrying diethylaminoethyl groups in an amount of 0.5 to 3 g per 1 kg of plasma, separated, followed by precipitation with ethanol to a final concentration de r solution of 9 vol .-% at -3 ° C, centrifuged the precipitate and dissolved in a citrate buffer at a pH of 6.35 and 37 ° C with stirring, the protein value of the fibrinogen solution with sodium citrate solution to 13.3 g / l and other accompanying proteins from 1 kg of fibrinogen solution with 260 ml of ethanol, 240 ml of glycine citrate buffer and 1500 ml of nitrate citrate solution, the precipitate is centrifuged off and the fibrinogen and the coagulation factor XIII from the supernatant by adding ethanol to a final concentration of Solution of 9 vol .-% at -3 ° C, the precipitate at pH 6.35 and 37 ° C with stirring in a citrate buffer to the desired fibrinogen concentration and the solution over a filter clarifier and a Millipore sterile filter filtered. 2. The method according to claim 1, characterized in that the desired fibrinogen concentration in the Plasma protein solution is in the range of 40 to 80 g / l.